Membrane fusion is a conserved process which is essential for cell growth, hormone secretion, and neurotransmission, and is vital to human health and pathophysiology. We have established the yeast vacuole as a technically advanced system for studying fusion mechanisms. Vacuole fusion occurs in stages: Priming is the ATP-dependent disassembly of SNARE complexes and the synthesis of phosphoinositides, tethering is the initial stage of vacuole adhesion, docking is the enrichment of fusion proteins and lipids into a fusion-dedicated microdomain which culminates in the association of proteins in trans, between the vacuoles, and hemifusion and fusion are the lipid rearrangements which permit compartment mixing. These are catalyzed by SNAREs which bind to each other, in cis or in trans, SNARE chaperones (Sec17p/18p), the Rab GTPase Ypt7p, regulatory lipids (sterol, diacylglycerol, and phosphoinositides), and the HOPS complex which interacts with each of these key elements and links their functions. We have studied fusion with the intact organelle, and have purified each key element and reconstituted their functions with liposomes. We now propose to: 1. Exploit our liposomal reconstitution of fusion to understand its mechanism, 2. Analyze the proteins and dynamics of trans-SNARE complexes, 3. Determine the factors which cause SNARE-mediated bilayer disruption to lead to fusion rather than lysis, and 4. Measure the affinities between fusion catalysts and rate constants for subreactions to lay the foundation for quantitative understanding o fusion. PUBLIC HEALTH RELEVANCE: Membrane fusion is fundamental to cell growth, hormone secretion, and neurotransmission, yet its molecular mechanisms remain unclear. We study membrane fusion with yeast vacuoles, and have defined the lipids and proteins which catalyze vacuole fusion, purified each, reconstituted authentic fusion with these pure proteins, and established fundamental outlines of fusion mechanisms. Our proposed studies will exploit this system to determine how the fusion catalysts and lipids work together to give orderly fusion while maintaining organelle integrity, a fundamental process for human health and disease.